Arf and HDM2 interaction domains and methods of use thereof

Abstract

The present invention discloses that the binding of Arf with Dm2 results in specific domains of both proteins undergoing a dramatic transition from disordered conformations to extended structures comprised of β-strands. The presence of these specific domains is necessary and sufficient for the formation of the highly stable extended β structures formed between these two proteins. The present invention further exploits this discovery by providing unique methods for identifying and/or designing compounds that mimic, inhibit and/or enhance the effect of Arf on Dm2. The present invention also provides specific protein fragments derived from Arf and Dm2 that play a critical role in the binding of these two important regulatory proteins.

What is claimed is:  
     
         1 . A method of identifying a compound that can induce the formation of β-strand assembly of Dm2 comprising: 
 (a) contacting the compound with Dm2 or an inducible fragment of Dm2; and  
 (b) determining whether Dm2 or the inducible fragment of Dm2 is induced to form a β-strand assembly; wherein a compound is identified when Dm2 or the inducible fragment of Dm2 is induced to form a β-strand assembly.  
 
     
     
         2 . The method of  claim 1  wherein said determining is performed by circular dichroism measurements.  
     
     
         3 . The method of  claim 1  wherein said determining is performed by nuclear magnetic resonance.  
     
     
         4 . The method of  claim 1  wherein said determining is performed by Fourier Transform Infra-red spectroscopy.  
     
     
         5 . The method of  claim 1  wherein said determining is performed by fluorescence spectroscopy.  
     
     
         6 . The method of  claim 5 , wherein said determining is performed by monitoring the fluorescence of a native tryptophan of Dm2 or of the inducible fragment of Dm2.  
     
     
         7 . The method of  claim 5 , wherein Dm2 or the inducible fragment of Dm2 is labeled with a fluorescent probe, and wherein said determining is performed by monitoring the fluorescence of the fluorescent probe.  
     
     
         8 . The method of  claim 1  wherein the Dm2 is Hdm2 having the amino acid sequence of SEQ ID NO:8.  
     
     
         9 . The method of  claim 1  wherein the inducible fragment of Dm2 comprises amino acid residues 235-259 of SEQ ID NO:8, the H1 segment.  
     
     
         10 . The method of  claim 9  wherein the inducible fragment of Dm2 further comprises amino acid residues 275-289 of SEQ ID NO:8, the H2 segment.  
     
     
         11 . The method of  claim 1  wherein the inducible fragment of Dm2 comprises amino acid residues 275-289 of SEQ ID NO:8, the H2 segment.  
     
     
         12 . A compound identified by the method of  claim 1;  wherein said compound is not a peptide comprising five or more consecutive amino acids comprised by a naturally occurring protein.  
     
     
         13 . A method of identifying a compound that can enhance the rate of β-strand assembly of Dm2 induced by Arf comprising: 
 (a) contacting the compound with Dm2 or an inducible fragment of Dm2, and Arf or an inducing fragment of Arf; and  
 (b) determining the rate of the β-strand assembly of Dm2 or of the inducible fragment of Dm2; wherein a compound is identified when the rate of the β-strand assembly of Dm2 or of the inducible fragment of Dm2 increases in the presence of the compound relative to in the absence of the compound.  
 
     
     
         14 . A method of identifying a compound that can inhibit the formation of β-strand assembly of Dm2 comprising: 
 (a) contacting the compound with Dm2 or an inducible fragment of Dm2, and Arf or an inducing fragment of Arf; and  
 (b) determining the rate of formation of a β-strand assembly of Dm2 or the inducible fragment of Dm2; wherein when the rate of formation of the β-strand assembly of Dm2 or the inducible fragment of Dm2 decreases in the presence of the compound relative to in its absence, the compound is identified as a compound that can inhibit the formation of β-strand assembly of Dm2.  
 
     
     
         15 . A method of identifying a compound that can inhibit the formation of β-strand assembly of Dm2 comprising: 
 (a) contacting the compound with Dm2 or an inducible fragment of Dm2, and Arf or an inducing fragment of Arf; and  
 (b) determining the amount of formation of a β-strand assembly of Dm2 or the inducible fragment of Dm2; wherein when the amount of formation of the β-strand assembly of Dm2 or the inducible fragment of Dm2 decreases in the presence of the compound relative to in its absence, the compound is identified as a compound that can inhibit the formation of β-strand assembly of Dm2.  
 
     
     
         16 . A method of identifying a compound that can induce the formation of supramolecular assemblies comprised of β-strands of Dm2 comprising: 
 (a) contacting the compound with Dm2 or an inducible fragment of Dm2; and  
 (b) determining whether Dm2 or the inducible fragment of Dm2 is induced to form supramolecular assemblies comprised of β-strands of Dm2; wherein when Dm2 or the inducible fragment of Dm2 is induced to form supramolecular assemblies the compound is identified as a compound that can induce the formation of supramolecular assemblies comprised of β-strands of Dm2  
 
     
     
         17 . The method of  claim 16  wherein said determining is performed by size exclusion determinations.  
     
     
         18 . The method of  claim 16  wherein the Dm2 is Hdm2 having the amino acid sequence of SEQ ID NO:8.  
     
     
         19 . The method of  claim 16  wherein the fragment of Dm2 comprises amino acid residues 235-259 of SEQ ID NO:8, the H1 segment.  
     
     
         20 . The method of  claim 19  wherein the fragment of Dm2 further comprises amino acid residues 275-289 of SEQ ID NO:8, the H2 segment.  
     
     
         21 . The method of  claim 16  wherein the fragment of Dm2 comprises amino acid residues 275-289 of SEQ ID NO:8, the H2 segment.  
     
     
         22 . A compound identified by the method of claim  16 ; wherein said compound is not a peptide comprising five or more consecutive amino acids comprised by a naturally occurring protein.  
     
     
         23 . A method of treating a patient with cancer or a predisposition for getting cancer comprising administering to the patient a compound that can induce β-strand assembly of Hdm2 in a cell.  
     
     
         24 . The method of  claim 23  wherein the patient has a tumor with cells that are characterized by a lack of sufficient Arf activity to induce cell cycle arrest and/or apoptosis; but wherein the cells retain functional p53.  
     
     
         25 . A kit for identifying a compound that can induce β-strand assembly of Dm2 comprising: 
 (a) a peptide comprising an amino acid sequence selected from the group consisting of amino acid residues 235-259 of SEQ ID NO:8; amino acid residues 275-289 of SEQ ID NO:8, and both amino acid residues 235-259 and amino acid residues 275-289 of SEQ ID NO:8; and  
 (b) a peptide that comprises two copies of the Arf motif comprising the amino acid sequence of SEQ ID NO:13.  
 
     
     
         26 . The kit of  claim 25  further comprising instructions for identifying a compound that can induce β-strand assembly of Dm2.  
     
     
         27 . An antibody raised against a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:13, amino acid residues 235-259 of SEQ ID NO:8, and amino acid residues 275-289 of SEQ ID NO:8.  
     
     
         28 . The antibody of  claim 26  that is a humanized antibody.  
     
     
         29 . A method of inducing apoptosis in a cell by administering the antibody of  claim 28  to the cell.  
     
     
         30 . A method of treating a patient having a tumor comprising administering the antibody of  claim 29  to the patient; wherein the tumor contains cells characterized by having functional Arf, functional Hdm2 and functional p53.  
     
     
         31 . A method of designing a compound that is predicted to mimic the ability of Arf to induce the formation of the β-strand assembly of Dm2, said method comprising: 
 (a) generating a computer model of a structure of an Arf-Dm2 complex based on: 
 (i) the amino acid sequence of the portions of Arf and Dm2 involved in the Arf-Dm2 complex; and  
 (ii) the circular dichroism and Fourier Transform Infra-red spectra obtained for the Arf-Dm2 complex; and  
 
 (b) designing a compound to bind to Dm2 as Arf does using the computer model of the structure of the Arf-Dm2 binding complex generated in step (a); wherein said compound is predicted to mimic the ability of Arf to induce the formation of the β-strand assembly of Dm2.  
 
     
     
         32 . The method of  claim 31 , further comprising: 
 (c) organically synthesizing said compound;    (d) contacting the synthesized compound with a Dm2 or an inducible fragment of Dm2; and    (e) determining whether the Dm2 or the inducible fragment of Dm2 has formed of a β-strand assembly; wherein when the Dm2 or the inducible fragment of Dm2 is induced to form a β-strand assembly in step (d), the synthesized compound is identified as a compound that mimics the ability of Arf to induce the formation of the β-strand assembly of Dm2.    
     
     
         33 . A peptide consisting of the amino acid sequence of SEQ ID NO:13.  
     
     
         34 . The peptide of  claim 33  consisting of the amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.  
     
     
         35 . A fusion protein comprising a peptide consisting of the amino acid sequence of SEQ ID NO:13.  
     
     
         36 . A peptide consisting of two segments of an Arf protein, wherein each segment consists of the amino acid sequence of SEQ ID NO:13.  
     
     
         37 . The peptide of  claim 36  wherein at least one segment consists of the amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.  
     
     
         38 . The peptide of  claim 37  wherein the other segment consists of the amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.  
     
     
         39 . A fusion protein comprising a peptide consisting of two segments of an Arf protein, wherein each segment consists of the amino acid sequence of SEQ ID NO:13.  
     
     
         40 . A peptide consisting of amino acid residues 235-259 of SEQ ID NO:8, the H1 segment.  
     
     
         41 . A fusion protein comprising a peptide consisting of amino acid residues 235-259 of SEQ ID NO:8.  
     
     
         42 . A peptide consisting of amino acid residues 275-289 of SEQ ID NO:8.  
     
     
         43 . A fusion protein comprising a peptide consisting of amino acid residues 275-289 of SEQ ID NO:8.  
     
     
         44 . A peptide consisting of amino acid residues 235-259 and amino acid residues 275-289 of SEQ ID NO:8.  
     
     
         45 . A fusion protein comprising a peptide consisting of amino acid residues 235-259 and amino acid residues 275-289 of SEQ ID NO:8.  
     
     
         46 . A composition comprising two segments of an Arf protein chemically joined via a non-peptide linkage, wherein each segment comprises the amino acid sequence of SEQ ID NO:13.